休止期纖維母細胞比增生期纖維母細胞具有更強的發炎反應

Quiescent fibroblasts are more active in mounting robust inflammatory responses than proliferative fibroblasts

陳柏叡(Bo-Rui Chen)

指導教授 ： 伍焜玉

國立清華大學/生命科學院/生物科技研究所/博士(2012年)

摘要

一般認為細胞在進入休止期(G0/G1 phase)時是不具有活動力且類似進入睡眠的狀態。而在我們實驗室先前的研究報告指出，休止期人類纖維細胞比增生期的細胞在有前發炎因子的刺激下會大量表現環氧化酵素二型 cyclooxygenase-2 (COX-2)。然而最新的研究報告更指出當細胞進入休止期時其細胞生理代謝的路徑上比增生期的細胞更是扮演了重要的角色。本論文的研究利用未添加血清培養細胞24小時使細胞進入休止期與添加血清培養細胞24小時
使細胞進入增生期，再使用前發炎因子刺激細胞4小時來促使COX-2表現。利用人類基因微陣列技術來分析休止期與增生期的纖維母細胞在前發炎因子的刺激後其人類基因的表現。另外使用藥物抑制劑來觀察休止期與增生期纖維母細胞訊息傳遞及轉錄機制的不同。藉由藥物型的抑制劑及基因靜默我們發現了蛋白激酶C protein kinase C δ (PKCδ)與胞外訊息調節激酶extracellular signal regulated protein kinase 1/2 (ERK1/2)調控著COX-2在休止期細胞裡的表現。然而當細胞進入增生期時，COX-2的表現卻無法被PKCδ 或 ERK1/2抑制劑所抑制，其原因在於增生期的細胞本身含有抑制PKCδ 和 ERK1/2表現的物質使得抑制劑效果不理想。在這個研究中也發現COX-2轉錄表現在增生期的細胞降低乃因為轉錄因子NF-κB的結合COX-2 promoter的能力變弱導致COX-2表現變低。在人類基因微陣列的實驗結果顯示出，有35個基因在休止期的細胞裡具有高度表現，而這些基因大多數屬於會被NF-κB轉錄因子所調控的前發炎性細胞激素、細胞激素、黏附分子及基質金屬蛋白酵素。在細胞遷移的實驗裡可發現休止期的纖維母細胞在佛波醇-12-肉蔻酯-13-乙酯(phorbol 12-myristate 13-acetate, PMA)的刺激下會促使細胞遷移，並且比增生期的纖維母細胞還具有高度的遷移能力。而選擇型的COX-2抑制劑可以抑制細胞的遷移能力並且被前列腺素前列腺素E二型給恢復其遷移能力。COX-2的大量表現及發炎因子的刺激會造成DNA的氧化，PMA在休止期的纖維母細胞裡可以顯著地的促使8-羥基去氧鳥糞核苷(8-OHdG)的增加。在這些研究的結果指出，休止期的纖維母細胞(或是其他的休止期細胞)在PMA的刺激後，會藉由前發炎因子基因PKCδ 和 ERK1/2訊息路徑而產生發炎反應並進而促使細胞遷移和造成大量的DNA氧化使得細胞受傷。而當纖維母細胞在進入增生期時，本身會釋放出可抑制COX-2表現的物質減少前發炎因子基因的表現並且抑制PKCδ 和 ERK1/2訊息傳遞路徑使得發炎反應及DNA氧化降低進而保護細胞。然而在以肺癌和乳癌細胞裡卻無法觀察到此現象，而這個研究也說明了COX-2的表現在正常的纖維母細胞裡與細胞週期表現是息息相關的。

關鍵字

休止期 ； 纖維母細胞 ； 環氧化酵素二型 ； 發炎反應 ； 蛋白激酶C δ ； 胞外訊息調節激酶

並列摘要

Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C δ (PKCδ) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKCδ or ERK1/2 inhibitors due to intrinsic inhibition of PKCδ and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-κB binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-κB for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E2. As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKCδ/ERK1/2 signaling pathway.

並列關鍵字

Quiescent phase ； fibroblast ； cyclooxygenase-2 ； inflammatory responses ； protein kinase C δ ； extracellular signal regulated protein kinase 1/2

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